HIGH-THROUGHPUT HTLV-1 PROVIRAL DNA DETECTION SYSTEM USING A NUCLEIC ACID EXTRACTION ROBOT AND REAL-TIME PCR DETECTION

A high-throughput nucleic acid testing system was developed for detecting proviral DNA of human T-cell leukemia virus type 1 (HTLV-1), using an automatic nucleic acid extractor and the real-time detection TagMan PCR (TagMan PCR) targeting the pX region of the HTLV-1 genome. Approximately 4μg and 2.5μg of DNA were obtained from 200μl of whole blood and 100μl of frozen blood cells separated from whole blood, respectively. Extraction of nucleic acid from 48 blood samples was completed within 120 minutes. The detection limit of the TagMan PCR was as high as that of the nested PCR. Amplification and detection of HTLV-1 genome in 96 blood samples was completed within 160 minutes. Extraction plus TagMan PCR for viral genome as well as enzyme immunoassay (EIA) and indirect immunofluorescence assay (IF) for HTLV-1-antibodies were performed to test 38 blood samples which were determined to be HTLV-1-antibody positive by the donor screening test using particle agglutination. The results of EIA and IF coincided well with those of the TagMan PCR, indicating that this detection system for HTLV-1 provirus DNA was useful for testing many samples in a short time with high sensitivity and specificity.